Purification of an alternate form of the alpha subunit of the glycoprotein hormones from bovine pituitaries and identification of its O-linked oligosaccharide.

Extracts of bovine anterior pituitary glands contain significant amounts of material with immunological properties similar to those of the common, alpha, subunit isolated from the pituitary glycoprotein hormones. Purification of this "free alpha-like" material and analysis show it to contain an additional site of glycosylation not present in the alpha subunit isolated from intact glycoprotein hormones. This additional oligosaccharide is O-linked to a threonine residue corresponding to threonine-43 of bovine lutropin-alpha. Carbohydrate analysis shows 1.7 mol of sialic acid, 0.8 mol of galactose and 0.9 mol of galactosamine/mol of oligosaccharide. A similar structure for the free alpha-like material as compared to bovine lutropin-alpha is evident from equal potency in an anti-lutropin-alpha radioimmunoassay, a similar amino acid composition and similar but not identical peptide maps. The free alpha-like material is distinct from lutropin-alpha in that the free alpha-material contains sialic acid and galactose, has a slightly higher apparent molecular weight, an increased negative charge, and will not reassociate with native lutropin-beta. Peptide maps of the tryptic peptides of the free alpha-like material show additional differences (other than the O-linked oligosaccharide) when compared to peptide maps of lutropin-alpha; thus additional modifications are probably present.